The recent advances in the field of biotechnology have required faster and more accurate techniques for recovery, purification and analysis of biological and biochemical substances, such as proteins. Chromatography is a commonly used purification technique in this field. In chromatography, two mutually immiscible phases are brought into contact. More specifically, the target molecule is introduced into a mobile phase, which is contacted with a stationary phase. The target molecule will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. The interactions exploit differences in the physical or chemical properties of the components in the sample. In liquid chromatography, a liquid sample, optionally combined with a suitable buffer constitutes the mobile phase, which is contacted with a stationary phase, known as a separation matrix. Usually, the matrix comprises a support to which ligands, which are groups capable of interaction with the target, have been coupled.
Separation matrices are commonly based on supports made from inorganic materials, such as silica, or organic materials, such as synthetic or natural polymers. The synthetic polymers, such as styrene and divinylbenzene, are often used for supports that exhibit some hydrophobicity, such as size exclusion chromatography, hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC). Further, the synthetic polymers are sometimes preferred over natural polymers, as they are easily made more rigid and pressure-resistant, resulting in supports that provide more advantageous flow properties.
The natural polymers, which are commonly polysaccharides such as agarose, have been utilised as supports of separation matrices for decades. Due to the presence of hydroxyl groups, the surfaces of the natural polymers are usually hydrophilic, giving essentially no non-specific interactions with proteins. Another advantage of the natural polymers, which is of specific importance in the purification of drugs or diagnostic molecules for internal human use, is their non-toxic properties. Agarose can be dissolved in water at increased temperature, and will then form a porous gel upon cooling to a certain temperature (the gelling point). On heating, the gel will melt again at a temperature (the melting point), which is usually considerably higher than the gelation point. The gelation involves helix-helix aggregation of the polysaccharide polymers, and is sometimes referred to as a physical cross-linking.
As mentioned above, the natural polymers are less rigid and pressure-resistant than synthetic polymers, and consequently methods have been developed for improvement thereof. For example, by varying the concentration of polysaccharide, the porosity of the support may be increased, resulting in improved target mass transport and increase of the area with which the target interacts during chromatography. Another essential parameter to consider is the flow properties of the support, for example in a packed bed of particulate separation matrix. When the mobile phase is forced through the bed, the back pressure will mainly be controlled by the interstitial channels between the particles. At low flow rates, the particles can be regarded as incompressible and then the back pressure increases linearly with the flow rate, with the slope depending on the particle size. As the flow rate increases, the particles may start to deform under the hydrostatic pressure, resulting in diminishing diameters of the interstitial channels and a rapidly increasing back pressure. At a certain flow rate, depending on the rigidity of the matrix, the bed will collapse and the back pressure approaches infinity.
The most commonly way to improve the rigidity and hence the flow properties of agarose is chemical cross-linking thereof. Such cross-linking takes place between available hydroxyl groups, and may be obtained by commonly known methods using e.g. epichlorohydrin.
U.S. Pat. No. 4,973,683 (Lindgren) relates to the cross-linking of porous polysaccharide gels, and more specifically to a method of improving the rigidity while minimising the non-specific interaction of a porous polysaccharide gel. The method involves providing an agarose gel and a reagent denoted “monofunctional”, which comprises a reactive group, such as a halogen group or an epoxide group, and a double bond. The reagent is bound to the gel via its reactive group; and the double bond is then activated into an epoxide or halohydrin, which is finally reacted with hydroxyl groups on the agarose to provide cross-linking.
U.S. Pat. No. 5,135,650 (Hjerten et al) relates to highly compressible chromatographic stationary phase particles, such as agarose beads, which are stated to be sufficiently rigid for HPLC and non-porous to the extent that they are impenetrable by solutes. More specifically, the Hjerten beads are produced from porous agarose beads, which are contacted with an organic solvent to collapse the porosity, after which the bead surfaces inside the collapsed pores are cross-linked to fix the pores in their collapsed state. Alternatively, the beads are produced by filling the pores with a polymerisable substance, which grafts to the pore surfaces, and performing graft polymerisation. One stated advantage of the invention disclosed is that a single stationary phase is effective at high pressures and yet can be used at low pressures. However, the use of solvents, especially such solvents as used in this patent, is in general avoided for health and safety reasons.
U.S. Pat. No. 6,602,990 (Berg) relates to a process for the production of a porous cross-linked polysaccharide gel, wherein a bifunctional cross-linking agent is added to a solution of polysaccharide and allowed to bind via its active site to the hydroxyl groups of the polysaccharide. A polysaccharide gel is then formed from the solution, after which the inactive site of the cross-linking agent is activated and cross-linking of the gel performed. Thus, the cross-linking agent is introduced into the polysaccharide solution, contrary to the above discussed methods wherein it is added to a polysaccharide gel. The bifunctional cross-linking agent comprises one active site, i.e. a site capable of reaction with hydroxyl groups of the polysaccharide, such as halides and epoxides, and one inactive site, i.e. a group which does not react under the conditions where the active site reacts, such as allyl groups. Thus, the present bifunctional cross-linking agent corresponds to the “monofunctional reagents” used according to the above-discussed U.S. Pat. No. 4,973,683 (Lindgren). Particles comprised of the resulting gel have been shown to present an improved capability of withstanding high flow rates and back pressures. A drawback with the U.S. Pat. No. 6,602,990 method is that bromine is required for the activation of the cross-linking agent.
U.S. Pat. No. 5,998,606 (Grandics) relates to a method of synthesising chromatography matrices, wherein cross-linking and functionalisation of a matrix takes place simultaneously. More specifically, double bonds provided at the surface of a polymeric carbohydrate matrix are activated in the presence of a metallic catalyst to cross-link the matrix and functionalise it with halohydrin, carboxyl or sulphonate groups. The double bonds are provided at the matrix surface by contact with an activating reagent, which contains a halogen atom or epoxide and a double bond. Thus, the U.S. Pat. No. 5,998,606 activating reagent corresponds to the U.S. Pat. No. 4,973,683 monofunctional reagent and the U.S. Pat. No. 6,602,990 bifunctional cross-linking agent.
Qi et al (Journal of Functional Polymers, Vol. 13, March 2000: “Preparation of Two Types of Immobilized Metal-Chelated Complex Affinity Membrane Chromatography Media”) describes how macro-porous cellulose affinity membranes are produced using cellulose filter paper as the matrix, going through alkali treatment, epoxidation activation, coupling with disodium iminodiacetate, and immobilizing Cu2′. In addition, agarose was covalently cross-linked onto the post-activation membranes to produce membranes that possess a sandwich-like structure.
US 2005/0220982 (Moya et al) relates to a method of forming polysaccharide structures such as beads, gel films and porous coatings on porous substrates by forming a room-temperature gel-inhibited solution of a polysaccharide, one or more gel-inhibiting agent(s) and a solvent such as water, heating the mixture until all the components are dissolved, cooling the mixture as a solution to about room temperature, forming a three dimensional structure with the solution and adding the structure to a gelling agent to form a polysaccharide gel. The gel-inhibiting agent is e.g. based on zinc, lithium or sodium salts. The coating of polysaccharide is stated to be thick enough to allow for diffusive flow to occur within the polysaccharide layer itself. A stated advantage of the described method is that the structures are formed at room temperature and with controlled gelling of the polymer with polysaccharide polymers that normally gel well above room temperature. It is also stated that the coating of surfaces is achieved without substantially blocking the pores with the polysaccharide. However, such operating at room temperature will require careful control of the agarose solution, which makes the overall process relatively time-consuming.
Thus, even though techniques are available for producing cross-linked polysaccharide separation matrices, different future applications will put different requirements on the matrix, resulting in an ongoing need of alternative methods.